Environmental Toxicology and Chemistry, Vol. 34, No. 11, pp. 2513–2522, 2015 # 2015 SETAC Printed in the USA

LATENT COGNITIVE EFFECTS FROM LOW-LEVEL POLYCHLORINATED BIPHENYL EXPOSURE IN JUVENILE EUROPEAN STARLINGS (STURNUS VULGARIS) ALEXANDER R.D. ZAHARA,y NICOLE L. MICHEL,z LEANNE M. FLAHR,x LEANNE E. EJACK,y and CHRISTY A. MORRISSEY*yz yDepartment of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada zSchool of Environment and Sustainability, University of Saskatchewan, Saskatoon, Saskatchewan, Canada xToxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada (Submitted 18 January 2015; Returned for Revision 15 April 2015; Accepted 21 May 2015) Abstract: Ecotoxicology research on polychlorinated biphenyl (PCB) mixtures has focused principally on short-term effects on reproduction, growth, and other physiological endpoints. Latent cognitive effects from early life exposure to low-level PCBs were examined in an avian model, the European starling (Sturnus vulgaris). Thirty-six birds, divided equally among 4 treatment groups (control ¼ 0 mg, low ¼ 0.35 mg, intermediate ¼ 0.70 mg, and high ¼ 1.05 mg Aroclor 1254/g body weight), were dosed 1 d through 18 d posthatch, then tested 8 mo to 9 mo later in captivity in an analog to an open radial arm maze. Birds were subject to 4 sequential experiments: habituation, learning, cue selection, and memory. One-half of the birds did not habituate to the test cage; however, this was not linked to a treatment group. Although 11 of the remaining 18 birds successfully learned, only 1 was from the high-dosed group. Control and low-dosed birds were among the only treatment groups to improve trial times throughout the learning experiment. Highdosed birds were slower and more error-prone than controls. Cue selection (spatial or color cues) and memory retention were not affected by prior PCB exposure. The results indicate that a reduction in spatial learning ability persists among birds exposed to Aroclor 1254 during development. This may have implications for migration ability, resource acquisition, and other behaviors relevant for fitness. Environ Toxicol Chem 2015;34:2513–2522. # 2015 SETAC Keywords: Persistent organic pollutants

Avian behavior

Cognition

Thyroid hormone disruption

Neurodevelopment

in many environments where they can affect wildlife health [10–12]. Neurological impairment has been associated with developmental PCB exposure, including neurotransmitter disruption [13], altered intracellular signaling processes, reduced synaptic function in the hippocampus and cerebellum [14,15], and impaired thyroid hormone function [16]. In mammals, developmental PCB exposure has been linked to various behavioral changes and cognitive deficits: hyperactivity in mice [17], reduced spatial learning ability in rats [18,19], and reduced learning and memory in some mouse genotypes [20]. Although thyroid function may be less susceptible to PCB disruption in birds than in mammals [21], adverse consequences associated with early developmental exposure have been reported. Reduced egg quality, low hatching success rates, abnormal nestling morphology, decreased nestling survival, and reduced posthatching weights are examples of consequences associated with developmental PCB exposure in songbirds [7,22–24]. Of the numerous behavioral changes associated with developmental PCB exposure, perhaps the most detrimental to avian wildlife include impairments to spatial learning and memory. Accurate spatial memory is essential to bird species like chickadees, nutcrackers, and jays that have evolved complex memory maps to recover thousands of food caches [25]. In gregarious bird species, learning and intelligence have been linked to social dominance and increased access to resources [26]. In the seminal study by Perdeck [27], flocks of adult European starlings were shown to rely heavily on their spatial memory to successfully navigate during secondary migration events. Spatial learning ability has also been linked with song bout length, an indicator of social rank and reproductive success [28]. Spatial memory and learning are

INTRODUCTION

Persistent behavioral effects caused by developmental exposure to endocrine-disrupting chemicals remain an important yet understudied facet of ecotoxicology. Sublethal embryonic or neonatal exposure to xenobiotics during critical developmental windows can have adverse consequences for an organism later in life [1,2]. Thyroid hormones are widely known for their role in regulating early neurological and cognitive development [3]. Evidence from several mammalian studies also shows that several compounds, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers, and perchlorate, exhibit thyroid hormone–disrupting properties that can impair brain development [4]. Because embryonic and juvenile life stages rely heavily on timed release of thyroid hormones for proper neurological and cognitive development [5], early exposure to thyroid hormone–disrupting chemicals such as PCBs could have long-lasting effects on behavior and learning ability, which may persist into adulthood. Introduced in the early 20th century but banned in North America by 1979, PCBs were widely used as hydraulic lubricants, plasticizers, dielectric fluids for transformers, adhesives, flame retardants, sealants, and pesticide extenders [6]. They bioaccumulate in the tissues of wildlife and can be transferred to developing young through lactation, placental transmission, egg formation, or dietary consumption [7,8]. Although monitoring programs report declines in tissue residues since the implementation of bans [9], high PCB residues persist * Address correspondence to [email protected] Published online 29 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3084 2513

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strongly linked to specific brain regions—namely, the hippocampus—in both mammals and birds [29]. In pigeons, experimental hippocampal lesions have been shown to impair spatial learning and increased flight times from unknown release sites to home roosts [30] or slowed responses in finding hidden food rewards [31]. Given the widespread exposure of wildlife to PCBs and related compounds, it is important to examine the latent effects of endocrine-disrupting chemicals in the context of an organism’s full life history [32]. The present study seeks to determine whether persistent behavioral effects are induced by early PCB exposure during development. At 8 mo to 9 mo after an 18-d nestling PCB exposure, European starlings (Sturnus vulgaris) were assessed on their ability to habituate to the test cage (habituation), their ability to complete a food-finding task rapidly and error-free over multiple trials (learning), the predominance of spatial versus visual color cues used in completing a task (cue selection), and their ability to retain the newly learned information (memory). We hypothesized that starlings that received higher PCB doses as nestlings would show greater impaired learning ability and would shift from spatial to color cues as the dominant form of information encoding in compensation for reduced hippocampal or other regional brain function. MATERIALS AND METHODS

Animal ethics

All animal procedures described were reviewed and approved by the University Committee on Animal Care (protocol 201100143). Dosing and husbandry

As part of a larger study conducted in 2012, 84 free-living European starlings were hatched in nest boxes at the University of Saskatchewan’s Goodale Research Farm (Saskatoon, Saskatchewan, Canada: 52830 23.1300 , –1068300 47.9000 ). Nestlings were dosed by oral gavage with 0 mg/mL (ppm, vehicle control), 50 mg/mL (low dose), 100 mg/mL (intermediate dose), or 150 mg/mL (high dose) of Aroclor 1254 (Supelco Analytical) in sunflower oil daily from 1 d to 18 d posthatch. Doses equated to exposures of 0 mg, 0.35 mg, 0.70 mg, and 1.05 mg Aroclor 1254/g body weight. For example, a 10-g nestling in the high-dose group would be administered 0.07 mL of 150 mg/mL Aroclor 1254 dosing solution, amounting to 1.05 mg Aroclor 1254/g body weight. The doses are known to be well within the range that birds, including European starlings, may be exposed to in the wild [2,22] and produced liver residues in a subset of euthanized day-19 starlings from each dose group (geometric means: controls ¼ 0.69 mg/g, low dose ¼ 13.1 mg/g, intermediate dose ¼ 13.3 mg/g, high dose ¼ 38.7 mg/g; L.M. Flahr, 2014, Master’s thesis, University of Saskatchewan, Saskatoon, Saskatchewan). Individual nestlings within each nest box were assigned to different treatment groups to randomize any heritability or nest effects (n ¼ 21/treatment group); therefore, clutch sizes larger than 4 included 2 birds of the same dose group. A maximum of 3 nestlings were taken from a nest box at a time, which prevented any interruption in parental feeding. After handling and dosing, nestlings were immediately returned to their nest boxes and the remaining nestlings dosed. On day 18, nestlings were brought into captivity and housed as a nest group in cages at the Animal Care Unit at the Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada,

A.R.D. Zahara et al.

under a 15:9-h light:dark cycle. Nestlings were fed an organic mixture of turkey starter crumbles, hard-boiled eggs, pureed carrots, and multivitamin powder (Hagen Prime) 6 to 8 times per day until they were able to feed independently on dry turkey starter (approximately 30 d of age). Birds were then grouphoused in a free flight colony room (5  3  3 m) for 2 mo before being randomly assigned to cages with 2 to 3 birds per cage and given access to food and water ad libitum. Beginning in September 2012, as part of a companion study [33], juvenile captive starlings were gradually exposed to a shorter photoperiod over a 6-wk period until they were maintained on a 9:15-h light: dark winter schedule. This schedule was maintained throughout the present study. Apparatus

A common method used to study spatial learning is to place subjects in a radial arm maze. A radial arm maze is a symmetrical multiarm maze wherein a single arm is baited with a food reward; learning the location of the food reward requires the use of spatial memory. Other tests for spatial learning are the Morris water maze for rodents [34] or a “dry version” for nonswimming animals [29]. For testing birds, analogs to radial arm mazes and dry Morris water mazes accommodate flight by placing hidden food equidistantly throughout an open room or cage and releasing birds from different locations [35,36]. This experimental setup has been used effectively in different avian models, including in passerines such as the zebra finch (Taeniopygia guttata) [37]. An experimental cage (1  1  0.9 m; Figure 1) was set up within the existing colony room to maintain the social requirements of the experimental birds. A pilot experiment indicated that most starlings would not habituate in the

Figure 1. Diagram of analog to open radial arm maze experimental cage used in habituation, learning, cue selection, and memory trials.

Cognitive effects of low-level PCBs in juvenile starlings

experimental cage if outside their familiar colony room and without conspecifics nearby. Home cages were arranged to surround the experimental cage such that no side was biased to other cages throughout the room. The bottom of the cage was open to the concrete floor, and the top was covered with transparent mesh fabric. Four 20  20 cm entrance doors (1 per side) were located at the bottom of the test cage. Feeder dishes (4.5 cm deep) of 4 different colors (purple, green, yellow, or white) were placed equidistantly on each side, approximately 15 cm to 20 cm from the top of the cage such that birds could not see the contents before approaching. A camera was fixed to the ceiling 2 m above the top of the experimental cage such that the entire cage was in the field of view. The camera was connected to a digital video recorder and video monitor kept in an adjacent room so that the trials could be viewed and recorded without observer interference. Procedure

Fifty-seven dosed starlings were retained in captivity until the ages of 8 mo to 9 mo old (juveniles). Of these 57 birds used in the original dosing study, 9 birds were randomly selected from each of the 4 treatment groups, for a total of 36 birds. Experimenters were blind to treatment group to avoid bias until the entire experiment was complete. Birds were habituated and tested once for learning, selection, and retention in 1 of 3 separate rounds (n ¼ 12 birds/round, 14 birds/round, and 10 birds/round) within a span of 2 mo, using identical procedures for each round. Preparation and habituation. One week before habituation trials, feeder dishes were placed in the home cages of experimental birds. The dishes were filled with 1 to 4 mealworms and rotated daily to ensure that birds were exposed to each dish color. This was done both to prevent neophobic behavior when exposed to the dishes and to begin the associative learning process. During the habituation trials, each of the 4 dishes was placed in the experimental cage and baited with mealworms as a food reward. A bird was released into the experimental cage and left for a maximum of 15 min, once a day, over a 5-d period. Birds were removed sooner than 15 min if fed from all 4 dishes. Dish color/cage side combinations were rotated daily, as was the side of entry. The goal of this stage was to encourage exploration in the test cage, thereby reducing neophobic reactions. Birds that fed from any dishes 3 d out of the 5 d were considered to have habituated to the experimental cage. It was assumed that all birds entering the learning experiment had associated the dishes as being baited with a food reward but without prejudice for any specific dish color or location. Learning experiment. Each bird was randomly assigned a unique dish color/cage side combination, which remained consistent throughout the experiment. For each bird, 1 dish was baited with a mealworm, while the other 3 remained empty. Learning was assessed based on a bird’s ability to associate the unique dish color/cage side combination with a food reward as indicated by trial time to find the correct baited dish and the number of visits to incorrect unbaited dishes (referred to as “incorrect choice”). To qualify as an incorrect choice, a bird would need to perch on an unbaited dish. Birds were fasted for 30 min to 90 min prior to each trial, per previous studies [26,38]. Each round of birds was tested up to 3 times/d for 4 d consecutively (for a maximum of 11 trials). Birds were placed by hand into the experimental cage from a predetermined side that rotated for each trial; the point of entry was kept consistent among birds relative to their baited dish

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(i.e., all birds entered below, opposite, left, or right of their baited dish for a given trial). Trial time began as soon as the bird left the experimenter’s hand and finished either when a bird fed from a baited dish or at a maximum of 300 s. A second experimenter observed the trial from a video monitor connected to a camera above the experimental cage, noting the number of incorrect choices and the trial start and end times. A bird was considered to have successfully completed the learning task after having found the food reward without error for at least 3 out of 4 consecutive trials within the time allowed. Trial outcomes were categorized as passing trials (no errors made), error trials, and nonattempt trials (bird did not attempt to feed). Once a bird completed the learning task without errors for 3 trials in a row, learning trials ended and a selection experiment was performed. For analytical purposes, birds that had successfully learned the maze before the 11th trial were given values of 0 for trial time and for incorrect choices in subsequent learning trials. Cue selection experiment. A cue selection experiment was conducted on birds that successfully completed the learning experiment to determine whether the birds were more dominantly relying on spatial or color cues when learning. To do so, a bird’s unique dish color and cage side were switched and both feeders were baited. We noted whether the birds chose the same feeder color on a different cage side (indicating the use of color cues) or chose a different feeder color on the same cage side (spatial cues). Trial time was not recorded for this experiment, and birds were tested only once. Memory retention experiment. Birds that completed the selection experiment were retested at 2 d and 5 d following the final selection trial under the same conditions as the learning experiment to determine their ability to retain learned information. This consisted of a single trial where the observer recorded time and any errors. Statistical analyses

The proportion of birds to successfully complete the habituation and learning tasks was analyzed by treatment group and sex using a Fisher exact test. General and generalized linear mixed models were constructed to evaluate the effects of PCB dose on trial time, probability of successful task completion, and probability of nonattempt trials. Fixed effects included PCB dose, sex, trial, and their interactions; subject, home cage, baited cage side, and baited dish color were included in the fully parameterized models as random effects. A Box-Cox transformation with l ¼ 0.16 was applied to the trial time data to reduce clustering in the residuals, and the model was analyzed using a generalized linear mixed model with a gaussian (normal) distribution using the package nlme [39]. The transformation and distribution were selected using Akaike information criterion, and the Box-Cox l was determined using the R package geoR [40]. Probability of successful task completion was analyzed using a generalized linear mixed model with a binomial distribution in the package lme4 [41]. We conducted exhaustive Akaike information criterion–based model selection to determine the fixed and random effects included in the final models [42]. Statistical significance was set at a ¼ 0.10 for all models a priori given the reduced power to detect effects using limited sample sizes in the various behavioral experiments. The mean learning rate, calculated as the slope of trial time over successive trials, was calculated using a general linear model with a random effect for individual birds using the package nlme.

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Cue selection and task completion frequencies were compared by treatment groups and sex using Fisher’s exact tests. Trial outcomes for the learning experiment (proportions of passing, error, and nonattempt trials) were compared by treatment group and sex with Pearson’s chi-squared tests. All analyses were conducted in R Ver 3.0.2 [43]. RESULTS

Habituation

Of the 36 birds originally placed in the test cage, half (n ¼ 18) successfully completed the habituation task. Slightly more females than males habituated to the test cage (Table 1), though the difference was not significant (p ¼ 0.18). Treatment group did not impact habituation ability, with nearly equal numbers of birds from each dose group successfully habituating to the test cage (Table 1). Learning

Task completion. For the learning trials, the number of birds from each treatment group to successfully complete the learning experiment varied. Of the 11 birds that learned the correct baited dish, only 1 (9%) belonged to the high-dose treatment group; the other 10 included 4 controls, 3 low-dose birds, and 3 intermediate-dose birds (Table 1). Five females and 2 males did not complete the learning task. Three of those females were high-dosed birds. Trial time. Overall, average trial time decreased throughout the 11 trials, indicating that most birds learned to find the correct baited dish over the course of the study (slope mean  standard error ¼ –5.6  1.7, p < 0.001; Figure 2A and Table 2). Trial time of both males and females significantly declined over repeated trials indicating learning occurred (Figure 2B and Table 2). However, prior exposure to Aroclor 1254 appeared to impair starling learning rates. Whereas trial time significantly declined over the course of the experiment in the control and low treatment groups, the intermediate and high treatment groups had slopes that were not significantly different from 0 (Table 2). Across all birds, trial time significantly declined over the course of the experiment (b ¼ –0.09  0.03, t ¼ –2.53, p ¼ 0.01; Table 3). High-dosed birds learned at a significantly slower rate than control birds: whereas trial time decreased over time in

Figure 2. Mean trial time for European starlings (Sturnus vulgaris) throughout the learning experiment by (A) treatment group and (B) sex. Control ¼ 0 mg Aroclor 1254/g body weight; low ¼ 0.35 mg Aroclor 1254/g body weight; intermediate ¼ 0.70 mg Aroclor 1254/g body weight; high ¼ 1.05 mg Aroclor 1254/g body weight. Error bars have been removed for visual clarity.

control birds, it remained relatively constant in birds exposed to the high Aroclor 1254 dose (significant treatment  trial interaction: b ¼ 0.078  0.045, t ¼ 1.75, p ¼ 0.08; Table 3). Trial times and learning rates were similar among males and females (Figure 2B and Table 3).

Table 1. Number of European starlings (Sturnus vulgaris) by treatment and sex to successfully complete each of the 4 experiments (habituation, learning, 2-d and 5-d retention, and cue selection) Dose treatment Experimental task a

Habituation DNC Learningb DNC Retention (2-d)c DNC Retention (5-d)c DNC Cue selection: Spatialc Cue selection: Colorc a

Control (n ¼ 9) 5 4 4 1 1 1 1 1 4 0

(56%) (44%) (80%) (20%) (50%) (50%) (50%) (50%) (100%) (0%)

Low (n ¼ 9) 4 5 3 1 3 0 2 1 1 2

(44%) (55%) (75%) (25%) (100%) (0%) (66%) (33%) (33%) (66%)

Intermediate (n ¼ 9) 4 5 3 1 2 0 2 0 2 1

(44%) (55%) (75%) (25%) (100%) (0%) (100%) (0%) (66%) (33%)

Sex High (n ¼ 9) 5 4 1 4 1 0 1 0 1 0

(56%) (44%) (20%) (80%) (100%) (0%) (100%) (0%) (100%) (0%)

p 1.00 0.27 0.63 1.00 0.20

Female (n ¼ 17) 11 6 6 5 4 0 4 0 5 1

(65%) (35%) (55%) (45%) (100%) (0%) (100%) (0%) (83%) (17%)

Male (n ¼ 19) 7 12 5 2 3 1 2 2 3 2

(37%) (63%) (71%) (29%) (75%) (25%) (50%) (50%) (60%) (40%)

p 0.18 0.64 1.00 0.43 0.55

All birds were tested in this experiment. Only habituated birds were tested in this experiment. c Birds that successfully passed the first 2 experiments (habituation and learning) were tested in this experiment. Control ¼ 0 mg Aroclor 1254/g body weight; low ¼ 0.35 mg Aroclor 1254/g body weight; intermediate ¼ 0.70 mg Aroclor 1254/g body weight; high ¼ 1.05 mg Aroclor 1254/g body weight; DNC ¼ did not complete (birds did not successfully pass the experiment). b

Cognitive effects of low-level PCBs in juvenile starlings

Environ Toxicol Chem 34, 2015

Table 2. Mean learning rate (slope of trial time across successive trials) of European starlings (Sturnus vulgaris) over the course of the study (n ¼ 18) by treatment group and sex

Grouping Treatment Control Low Intermediate High Sex Male Female Total

Table 4. Number and percentage of each trial outcome (passing, error, nonattempt) for European starlings (Sturnus vulgaris) by treatment group and sexa Group

Learning rate n

Slope  SE

t

df

p

5 4 4 5

–11.8  2.7 –10.1  3.8 –2.7  3.5 1.9  3.3

–4.3 –2.6 –0.8 0.6

49 39 39 49

Latent cognitive effects from low-level polychlorinated biphenyl exposure in juvenile European starlings (Sturnus vulgaris).

Ecotoxicology research on polychlorinated biphenyl (PCB) mixtures has focused principally on short-term effects on reproduction, growth, and other phy...
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